Capsid Region Research Articles

Complete Genome of a Novel Porcine Astrovirus

Astroviruses have been widely described in mammalian and avian species. Here, we report a complete genome sequence of a novel porcine astrovirus (PoAstV) isolated from a porcine fecal sample in China. The genome consists of 6,611 nucleotides, excluding the 3' poly(A) tail, and has two open reading frames (ORFs). ORF1 maps between nucleotide positions 19 and 4211 and encodes a 1,396-amino-acid (aa) polyprotein precursor consisting of nonstructural protein and putative RNA-dependent RNA polymerase, and ORF2 maps between nucleotide positions 4202 and 6531 and encodes a 775-aa polyprotein which is a capsid precursor protein. The genome sequence of the virus was distinct enough from those of the known PoAstVs to be considered a novel sequence. Phylogenetic analysis based on the predicted amino acid sequence of the complete capsid region showed that this strain may be a novel porcine astrovirus.

Novel Human Enterovirus C Infection in Child with Community-Acquired Pneumonia

Novel Human Enterovirus C Infection in Child with Community-Acquired Pneumonia

Poliovirus Evolution: The Strong, Silent Type

The high mutation rates of poliovirus and other RNA viruses promote viral diversity by rapid sampling of mutations. In this issue, Lauring et al. (2012) report that underlying synonymous codon variation in virus populations can have a considerable impact on adaptive potential with implications for viral fitness and virulence.

Characterization of non-polio enterovirus isolates from acute flaccid paralysis children in Pakistan reflects a new genotype of EV-107

Enteroviruses comprise a group of genetically diverse RNA viruses that usually cause respiratory illnesses, myocarditis, enteritis, meningitis, encephalitis and poliomyelitis. In this study, 23 non-polio enteroviruses, isolated during September–October 2009 from stool specimens of children suffering from acute flaccid paralysis (AFP) were subjected to serotype determination by using RIVM antiserum pools. Twenty-two isolates were identified as members of human enterovirus B (HEV-B) species except one that remained untypeable. Phylogenetic analysis of the untypeable isolate (PAKNIH-2009/23) based on the sequence of 5′UTR, capsid and 2A regions confirmed that this isolate is closely related to TN94-0349 strain of EV-107 serotype with 70.4% nucleotide similarity in VP1/2A junction, 79.4% in the capsid coding region and 89.1% in 5′UTR region respectively. Our data strongly suggests that PAKNIH-2009/23 is a genetically distinct strain of EV-107 currently circulating in Pakistan. This study also highlights the probable role of non-polio viral etiologies associated with AFP and needs to plan new strategies especially during the post polio eradication era.

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3 For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate. For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate. For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

Mechanical elasticity as a physical signature of conformational dynamics in a virus particle

In this study we test the hypothesis that mechanically elastic regions in a virus particle (or large biomolecular complex) must coincide with conformationally dynamic regions, because both properties are intrinsically correlated. Hypothesis-derived predictions were subjected to verification by using 19 variants of the minute virus of mice capsid. The structural modifications in these variants reduced, preserved, or restored the conformational dynamism of regions surrounding capsid pores that are involved in molecular translocation events required for virus infectivity. The mechanical elasticity of the modified capsids was analyzed by atomic force microscopy, and the results corroborated every prediction tested: Any mutation (or chemical cross-linking) that impaired a conformational rearrangement of the pore regions increased their mechanical stiffness. On the contrary, any mutation that preserved the dynamics of the pore regions also preserved their elasticity. Moreover, any pseudo-reversion that restored the dynamics of the pore regions (lost through previous mutation) also restored their elasticity. Finally, no correlation was observed between dynamics of the pore regions and mechanical elasticity of other capsid regions. This study (i) corroborates the hypothesis that local mechanical elasticity and conformational dynamics in a viral particle are intrinsically correlated; (ii) proposes that determination by atomic force microscopy of local mechanical elasticity, combined with mutational analysis, may be used to identify and study conformationally dynamic regions in virus particles and large biomolecular complexes; (iii) supports a connection between mechanical properties and biological function in a virus; (iv) shows that viral capsids can be greatly stiffened by protein engineering for nanotechnological applications.

Complete Genome Sequence of a New-Genotype Porcine Norovirus Isolated from Piglets with Diarrhea

Noroviruses (NoVs) are members of the family Caliciviridae and are emerging enteric pathogens of humans and animals. So far, porcine NoVs have been detected exclusively in fecal samples from adult swine without clinical signs. Here we report the genome sequence of a NoV strain isolated from piglets with diarrhea. Experimental infection of miniature pigs with this porcine NoV-positive fecal sample confirmed that this strain can cause diarrhea in piglets. A phylogenetic tree based on the predicted amino acid sequence of the complete capsid region showed that this strain is separate from known porcine GII strains (GII-11, GII-18, and GII-19), constituting the sole member of a new branch.

A Single Mutation in the Glycophorin A Binding Site of Hepatitis A Virus Enhances Virus Clearance from the Blood and Results in a Lower Fitness Variant

Hepatitis A virus (HAV) has previously been reported to bind to human red blood cells through interaction with glycophorin A. Residue K221 of VP1 and the surrounding VP3 residues are involved in such an interaction. This capsid region is specifically recognized by the monoclonal antibody H7C27. A monoclonal antibody-resistant mutant with the mutation G1217D has been isolated. In the present study, the G1217D mutant was characterized physically and biologically in comparison with the parental HM175 43c strain. The G1217D mutant is more sensitive to acid pH and binds more efficiently to human and rat erythrocytes than the parental 43c strain. In a rat model, it is eliminated from serum more rapidly and consequently reaches the liver with a certain delay compared to the parental 43c strain. In competition experiments performed in vivo in the rat model, the G1217D mutant was efficiently outcompeted by the parental 43c strain. Only in the presence of antibodies reacting specifically with the parental 43c strain could the G1217D mutant outcompete the parental 43c strain in serum, although the latter still showed a remarkable ability to reach the liver. Altogether, these results indicate that the G1217D mutation induces a low fitness phenotype which could explain the lack of natural antigenic variants of the glycophorin A binding site.

Multiple Antigenic Sites Are Involved in Blocking the Interaction of GII.4 Norovirus Capsid with ABH Histo-Blood Group Antigens

Noroviruses are major etiological agents of acute viral gastroenteritis. In 2002, a GII.4 variant (Farmington Hills cluster) spread so rapidly in the human population that it predominated worldwide and displaced previous GII.4 strains. We developed and characterized a panel of six monoclonal antibodies (MAbs) directed against the capsid protein of a Farmington Hills-like GII.4 norovirus strain that was associated with a large hospital outbreak in Maryland in 2004. The six MAbs reacted with high titers against homologous virus-like particles (VLPs) by enzyme-linked immunoassay but did not react with denatured capsid protein in immunoblots. The expression and self-assembly of newly developed genogroup I/II chimeric VLPs showed that five MAbs bound to the GII.4 protruding (P) domain of the capsid protein, while one recognized the GII.4 shell (S) domain. Cross-competition assays and mutational analyses showed evidence for at least three distinct antigenic sites in the P domain and one in the S domain. MAbs that mapped to the P domain but not the S domain were able to block the interaction of VLPs with ABH histo-blood group antigens (HBGA), suggesting that multiple antigenic sites of the P domain are involved in HBGA blocking. Further analysis showed that two MAbs mapped to regions of the capsid that had been associated with the emergence of new GII.4 variants. Taken together, our data map antibody and HBGA carbohydrate binding to proximal regions of the norovirus capsid, showing that evolutionary pressures on the norovirus capsid protein may affect both antigenic and carbohydrate recognition phenotypes.

Detection of Multiple Genotypes of Calicivirus Infection in Asymptomatic Swine in Taiwan

Noroviruses (NoVs) and sapoviruses (SaVs) of the family Caliciviridae are emerging enteric pathogens in humans and animals. Recent detection of genogroup II norovirus (GII NoV) RNA from swine raises public health concerns about zoonotic transmission of porcine NoVs to humans. However, few papers reported genotype distributions and epidemiological features in swine farms and their genetic relationship to human strains, which was the objective of our study. This study investigated the epidemiological features and genotypes of caliciviruses in swine farms using 533 pig faecal samples from six farms in central and southern Taiwan, tested for viral RNA using RT-PCR targeting the conserved polymerase gene. NoVs and SaVs were detected with a positive rate of 7.1% and 0.6%, respectively. To confirm the positive rate of NoVs, 255 pig faecal samples from two farms in central Taiwan were tested with primer pairs targeting the partial capsid gene of GII, and 32.3% of the positive rate was found. Furthermore, the results from the capsid region suggested a higher positive rate of 41.7% in winter than 26.4% in summer with statistical significance (P < 0.05). Sequence analysis showed 29 strains belonging to GII.4 (human) and nine strains belonging to GII.11 (swine) identified based on the partial polymerase gene. Additional genotypes clustered with GII.2 (human) and GII.18 (swine) were also characterized based on the partial capsid gene. SaVs detected in porcine faecal samples belonged to genogroup III (GIII), which clustered with the PEC-Cowden strain. Our study demonstrated the presence of multiple genotypes of both human and porcine NoVs infecting swine of various ages asymptomatically. Although the zoonotic potential of detected human NoVs in swine was not conclusive owing to the lack of local human faecal samples, our study revealed the importance of monitoring emerging strains in swine to mitigate the potential impact of recombinant NoVs infecting the human population.

Capsid protein sequence diversity of chicken astrovirus

The complete capsid gene sequences of 24 chicken astroviruses (CAstVs), collected in the UK, Germany, the Netherlands and South Africa from the 1980s to 2008, were determined and compared with that of a US CAstV (UGA-2006). Pairwise comparisons and phylogenetic analysis demonstrated the existence of two major capsid groups, designated A and B, which shared 38 to 40% amino acid identity. CAstVs from groups A and B shared capsid protein identities ranging from 26 to 38% with other avian astroviruses. The group A CAstVs comprised three subgroups, which displayed inter-subgroup identities ranging from 77 to 82%, while group B comprised two clearly separated subgroups, Bi and Bii, which displayed intra-subgroup identities of 97 to 99% and 94 to 99%, respectively, and shared inter-subgroup identities of 84 to 85%. Phylogenetic analyses performed with contiguous open reading frame 1b (polymerase) and open reading frame 2 (capsid) CAstV sequences showed that CAstVs from capsid subgroup Bi had polymerase genes that differed from those possessed by CAstVs belonging to group A and subgroup Bii. The N-terminal capsid regions (residues 1 to 415) were more conserved than the C-terminal regions, with the C-terminal regions of the subgroup Bi and Bii CAstVs sharing 76 to 78% amino acid identity, while the C-terminal regions of the A subgroups displayed identities less than 75%. CAstVs representative of both capsid groups and more than one subgroup were detected within the same broiler flock. The high level of capsid sequence diversity observed in this study has important implications for both the control and diagnosis of CAstV infections.

Emergence of antigenic variants with in serotype A foot and mouth disease virus in India and evaluation of a new vaccine candidate panel

Emergence of genetically and antigenically divergent lineages/genotypes and poor intergenotypic antigenic coverage is a major concern in serotype A foot-and-mouth-disease virus (FMDV) in India. In 2009, to cover antigenic diversity emerged in serotype A virus field isolates, IND40/2000 was selected as the new vaccine strain for incorporation in the trivalent FMD vaccine formulation used in India. Although current vaccine strain (IND40/2000) covers most isolates antigenically, a few VP359-deletion group isolates showed low r-value in routine vaccine matching exercise. The VP359-deletion group within genotype 18 emerged first in late part of 2002 and in 2007 causing outbreaks along with non-deletion isolates of the same genotype. In case of emergence or re-emergence of more antigenically divergent isolates in future, a need for a new vaccine candidate to cover maximum isolates of both deletion and non-deletion group may arise. Four alternate candidate vaccine strains (IND281/2003, IND195/2007, IND360/2007 and IND123/2008) were selected based on set criteria and antigenic relationships with field isolates sampled between 2002 and 2009 were analyzed using a micro-neutralization test. Phylogenetic analysis based on capsid region of serotype A isolates revealed existence of two broad distinct clusters (VP359-deletion and non-deletion group) within genotype 18. The VP359-deletion group has diversified genetically with time giving rise to three different sub-lineages (clade18a, 18b and 18c). The present study indicates that the virus candidates IND281/2003 (VP359-deletion group) and IND195/2007 (non-deletion group) can be used as an adjunct or alternative strain to currently used vaccine strain IND40/2000 in case of emergence of more antigenically divergent isolates in future.

Development and Validation of Novel AAV2 Random Libraries Displaying Peptides of Diverse Lengths and at Diverse Capsid Positions

Libraries based on the insertion of random peptide ligands into the capsid of adeno-associated virus type 2 (AAV2) have been widely used to improve the efficiency and selectivity of the AAV vector system. However, so far only libraries of 7-mer peptide ligands have been inserted at one well-characterized capsid position. Here, we expanded the combinatorial AAV2 display system to a panel of novel AAV libraries, displaying peptides of 5, 7, 12, 19, or 26 amino acids in length at capsid position 588 or displaying 7-mer peptides at position 453, the most prominently exposed region of the viral capsid. Library selections on two unrelated cell types-human coronary artery endothelial cells and rat cardiomyoblasts-revealed the isolation of cell type-characteristic peptides of different lengths mediating strongly improved target-cell transduction, except for the 26-mer peptide ligands. Characterization of vector selectivity by transduction of nontarget cells and comparative gene-transduction analysis using a panel of 44 human tumor cell lines revealed that insertion of different-length peptides allows targeting of distinct cellular receptors for cell entry with similar efficiency, but with different selectivity. The application of such novel AAV2 libraries broadens the spectrum of targetable receptors by capsid-modified AAV vectors and provides the opportunity to choose the best suited targeting ligand for a certain application from a number of different candidates.

Genetic characterization of vaccine and field strains of serotype A foot-and-mouth disease virus from India

Extreme antigenic and genetic heterogeneity of serotype A foot-and-mouth disease virus (FMDV) population has resulted in change of vaccine strains in India twice in the last decade. In such a situation, complete characterization of the vaccine strains is imperative. With regard to the frequent outbreaks of this disease, FMDV field strains are also of interest. Therefore three vaccine strains and two field strains of type A FMDV from India were completely sequenced and the obtained sequences were subjected to sequence and phylogenetic analyses. Based on the complete coding region, all the Indian strains clustered in the Asia topotype and exhibited a more than 11% nt divergence from the other Asian strains. The 5'-UTR of some Indian strains revealed block deletions of 43 and 86 nt corresponding to the pseudoknot region. Amino acids S44 in VP2 and F164 in VP1 were found to be the exclusive signatures for the Asia topotype. The vaccine strains differed at 65 aa positions in the capsid region, 13 of them antigenically critical. Variability at such positions is likely to affect the antigenic profile of these strains. Complete genome sequences of the vaccine strains presented here could serve as the reference for any comparative genomics in future.

Prevalence of Norovirus Infection in Children and Adults with Acute Gastroenteritis, Tehran, Iran, 2008–2009

Noroviruses are one of important agents that cause acute viral gastroenteritis worldwide. These viruses are belonging to Caliciviridae family and are genetically diverse. To date, there is no valuable data about prevalence of norovirus infection and the dominant genogroup/genotype among Iranian population. The objective of this study was to determine the frequency of norovirus infection in Iranian patients with gastroenteritis referred to three hospitals of Tehran and to specify the dominant genogroup/genotype of this virus among our study population. A total of 293 patients with acute gastroenteritis were included in the study. Detection of norovirus was performed using RT-PCR method and confirmed by direct sequencing with specific designed primers for capsid region of norovirus genome. Phylogenetic analysis was performed using the neighbor-joining method. Norovirus strains identified in our study were subsequently categorized according to previously defined genogroup/genotypes. Of these, norovirus GII was dominant genogroup. Sixty-five percent (17 of 26) of positive samples were determined as GII and 35% (9 of 26) were determined as GI, respectively, in 2008-2009. And among 8 sequenced strains of genogroup II the most frequent genotype was GII.3. The results of this study indicated that norovirus must be considered as one of the infectious causes of acute gastroenteritis among Iranian population. We also found that GII.3 is more prevalent in our study population. To the best of our knowledge there is limited data about the role of noroviruses in children and adults' acute gastroenteritis among Iranian patients and this prevalence and genotyping report of norovirus infection could be remarkable for further studies.

Mutations in VP2 and VP1 capsid proteins increase infectivity and mouse lethality of enterovirus 71 by virus binding and RNA accumulation enhancement

Enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease. EV71 infection occasionally associates with severe neurological sequelae such as brainstem encephalitis or poliovirus-like paralysis. We demonstrated that mouse-adapted strain increases infectivity, resulting in higher cytotoxicity of neuron cells and mortality to neonatal mice than a non-adapted strain. Results pointed to EV71 capsid region determining viral infectivity and mouse lethality. Mutant virus with lysine to methionine substitution at VP2149 (VP2149M) or glutamine to glutamic acid substitution at VP1145 (VP1145E) showed greater viral titers and apoptosis. Synergistic effect of VP2149M and VP1145E double mutations enhanced viral binding and RNA accumulation in infected Neuro-2a cells. The dual substitution mutants markedly reduced value of 50% lethal dose in neonatal mice infection, indicating they raised mouse lethality in vivo. In sum, VP2149M and VP1145E mutations cooperatively promote viral binding and RNA accumulation of EV71, contributing to viral infectivity in vitro and mouse lethality in vivo.

Surveillance of hepatitis A and E viruses contamination in shellfish in Thailand

To survey for hepatitis A virus (HAV) and hepatitis E virus (HEV) contamination in edible bivalve shellfish. A total of 213 shellfish (52 oysters, 69 cockles and 92 mussels) collected from a culture farm and two retailed markets were investigated for HAV and HEV contamination by reverse transcription-polymerase chain reaction (RT-PCR) assay using HA2-HA1 (capsid region) and HE366-HE363 (ORF2/3 overlapping region) primers, respectively. It was found that 3.8% of the shellfish and 2.9 and 6.5% of the cockle and mussel, respectively, showed positive for HAV detection. Nucleotide sequencing of all the 8 HAV-positive shellfish revealed 97-100% similarity to HAV subgenotype IA. Interestingly, viruses were found more frequently in the gills than in digestive tissue (4.5%vs 0.5%, P = 0.045). All the shellfish were negative for HEV. Significant contamination of HAV in edible bivalve shellfish was observed. Beside digestive tissue, gills are one of the important samples for viral genome detection. HAV-contaminated shellfish can play a role as reservoirs and/or vehicles in faecal-oral transmission in Thailand, and further monitoring of such a contamination is required.

Limited evidence of human papillomavirus on breast tissue using molecular in situ methods

Human papillomavirus (HPV) has been proposed as an etiologic agent of breast cancer based on numerous reports of high-risk (oncogenic) HPV types in malignant breast tissues. However, most of those studies used standard and nested solution polymerase chain reaction (PCR) techniques, both of which are disadvantaged by vulnerability to laboratory contamination from positive control DNA and the inability to localize the signal to a specific cell type. To overcome these drawbacks, the authors of this report explored the use of in situ molecular methods of viral detection to reassess the frequency of HPV in malignant breast tissue. In situ hybridization (ISH) was used with probes that were specific for the capsid region of 12 oncogenic HPV types, and in situ PCR (IS-PCR) was used with primers that were specific for the capsid region of HPV-16, which is the most common oncogenic HPV type. These methods were resistant to molecular contamination and allowed identification of the positive cell type. The specimens examined were malignant tissues from patients with 70 breast cancer patients at The University of Texas M. D. Anderson Cancer Center in Houston, Texas. HPV was observed in 4 of 70 specimens (5.7%) using ISH and in 2 of 70 specimens (2.9%) of specimens using IS-PCR. Concordance between the 2 methods was high for negative specimens; both methods yielded negative results in 66 of 70 specimens (94.3%). However, there was no concordance for the few positive specimens, probably because of differences in sensitivity and the targeted HPV types. Oncogenic (high-risk) HPV types were present in malignant breast epithelium very infrequently and, thus, may be causative agents of only a relatively small proportion of all breast cancers.

Novel Surveillance Network for Norovirus Gastroenteritis Outbreaks, United States

CaliciNet, the outbreak surveillance network for noroviruses in the United States, was launched in March 2009. As of January 2011, twenty state and local health laboratories had been certified to submit norovirus sequences and epidemiologic outbreak data to CaliciNet. During the network's first year, 552 outbreaks were submitted to CaliciNet, of which 78 (14%) were associated with foodborne transmission. A total of 395 (72%) outbreaks were typed as GII.4, of which 298 (75%) belonged to a new variant, GII.4 New Orleans, which first emerged in October 2009. Analysis of the complete capsid and P2 region sequences confirmed that GII.4 New Orleans is distinct from previous GII.4 variants, including GII.4 Minerva (2006b).